Streptonigrin antibiotics and process for their production



March 5, 1968 w. s. MARSH ET AL 3,372,090

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION 7 Sheets-Sheet 1 Filed July 5, 1963 SH S R mm V m8 M m L m wz0mo=z Z- IhOZMJ m 3 W E 2 m. u. o m v w h w m e m N C.- M 0m M W M S N A R T T N E m E 0 0 P Omw 00h 00m 00m 000- OO: OON 00 2 O0! 002 OOON OODN 000m 80 HEW W E N R O n A STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION '7 Sheets-Sheet 2 Filed July .5, 1963 PERC ENT TRANSMITTANCE wzomoi z Ewzw m s N Z O- m m h w 0 Q n 00m 000- 00: OON OOQ 00. 00m; OOON OOnN 000m 000? N INVENTORJ 0 WILLIAM S.MARSH ALINE L.GARRETSON KOPPAKA v. RAO

' ATTORNEYS PERCENT TRAN SMlTTANCE March 5, 1968 w. s. MARSH ET AL 3,372,090

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION Filed July 5, 1963 '7 Sheets-Sheet 3 o PERCENT TRANSMITTANOCE N Z n: 2 z o a. m m I 2 E:- E 8 9 O m o 9 0: LL 2 o E o E a E U) 9 E 3 I m D: LIJ 0 iii 0 lm o 5- o E Q 2 FJQ- 2 O on rs, w U Q LU o z 3 g E g a: O P. w m w a LL! 2 o O D: m 3 Z Q 0 0 r0 0 P1) 0 0 7 G N INVENTORS a; V9 v m 0 WILLIAM SMARSH PERCENT TRANSMITTANCE UNE LGARRETSON KOPPAKA V. RAO

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W ATTORNEYS March 5, 1968 w 5, MARSH ET AL 3,372,090

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION 7 Sheets-Sheet 4 Filed July 6, 1965 S N RH O EA E RR V AV W L K A E L N W l ILO wzomuzz z zkozm w z wAK O m N. O. m m b w m w m N E E C N N A A T T T n W M S S N N A M mow T T T N N E E om C R R E E P P OO- omm 00m 000- OOON 00mm DOOM 80 000m OO:OON OOn O21 002 E 0 Z m m m m E D z m 4 W O m March 5, 1968 w. s. MARSH ET AL 3,372,090

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION Filed July 5, 1963 7 Sheets-Sheet 5 PERCENT TRANSMITTANCE' o o O o i '2 E DC 9 2 Z a: 9. o 0. w 8 E m w 2 LL 0 D: o 1 3 2 2 z 2 9 2 D U a: g o E 0 5 g g m I M w 0 o. 8 o

Z 0 m L O i w I a z E D: 3 3 O o 3 3 O (D UJ O: 8 CC 2 3 O 8 r N 0 I0 0 O Q Q g N INVENTORS m WILLIAM S.M'ARSH PERCENT TRANSWTTANCE ALINELGARRETSON KOPPAKA V. RAO

ATTORNEYS March 5, 1968 w, s MARSH ET AL 3,372,090

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION Filed July 5, 1963 7 Sheets-Sheet e PERCENT TRANSMITTANCE Q o o 9 w t N O N Q. E O: (D g 8 O a) 55 n: 8 lw (n 2 u o O E 8 o 5 2 0 E D Z O: Z lw 8 U D: I. m I

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N INVENTORS 0 WILLIAM S. MARSH PERCEN TRANSMITTANGE ALINE LGARRETSON vKOPPAKA V. RAO

March 5, 1968 w. s. MARSH ET AL 3,372,099

STREPTONIGRIN ANTIBIOTICS AND PROCESS FOR THEIR PRODUCTION '7 Sheets-Sheet '7 Filed July 5, 1963 PERCENT TRANSMITTANCE OOm 00m 002 O0:

ZEQZOFQMEPw m0 mmkmw 4 IPm m0 EDIPQMEm zOEhEOmm omm mmz INVENTORS WILLIAM S.MARSH PERCENT TRANSMITTANCE ALINE LGARRETSON KOPPAKA V. RAG

ATfORNEYS @W/ 0 m. W

BY A g saunas Patented Mar. 5, 1968 3,372,090 tively the spectra of streptonig-rin P-1 and P-2, and FIG- STREPTONIGRIN ANTIBIGTICS AND PROCESS URE VII illustrates the spectrum of the methyl ester of FOR THEE PRODUCTION streptonigrin. Wlulam Marsh Wanaque, Ganeiso! Rlve' The newly discovered strains of microorganisms which Edge, and Koppaka V. Rao, Pinebroolr, N.J., assignors 5 produce streptonigrin and related compounds and which to Chas. Pfizer & Co., Inc, New York, NY, a are employed in the valuable process of the present inporation of Delaware Continuafi0n in part of applicafiens 753,967, vention were isolated from soil samples on agar formula- Aug. 8, 1958, Ser. No. 823,459, June 29, 1959, Ser. tlons- No, 39,535, J 29, 1960, d 8 N 190,520, Their identification 1n the culture collection of Chas.

Apr. 17, 1962. This application July 5, 1963, Ser. No. 1 0 Pfizer & Co., Inc. and the numbers assigned to them by 293,148 the American Type Culture Collection in Washington,

13 Clalms- DC. where they have been deposited are given below: This invention relates to the cultivation under con- Chm Pfi & trolled conditions of newly discovered strains of microoridentification; ATCC ganisms, to the biologically active substances produced 15 Isolate 3 streptomyces flocculus 1 5 5 thereby, to the metal salts and other derivatives of these Isolate BA 163 Smpmmyces flocculus 57 substances, and to methods for the recovery and concen- Isolate B A4721, Strepwmyices flacculus 13536 tration from crude solutions, including fermentation Isolate B A 13023 streptomymes born-opens 13854 broths, of these biologically active substances, particularly Isolate BA 16986 streptomyces fungicidicus 13853 of that substance hereinafter named streptonigrin. This Isolate 34 5 Strepmmyces flocculus 13 52 application is a continuation-in-part of our earlier filed Isolate 5 streptomyces flocculus 13851 copending US. applications Ser. No. 753,967, filed Aug. 8, Isolate 35412 streptomyces flocculus 13350 1958, Ser. No. 823,469, fi-led June 29, 1959, Ser. No. Isolate 35599 Streptomyces flow-alas 13359 39,535, filed June 29, 1960, and Se-r. No. 190,520, filed Isolate BA 89818, Strepmmyces flocculus 13855 Apr. 17, 1962, all now abandoned. Isolate BA-90792, Streptomyces flodculus 13860 This invention relates specifically to streptonigrin and {some 9079 Streptornyces flocculus 13357 to closely related substances, referred to generally herein I l t BA. 90926, Streptomyces flocculus 13861 as streptonigrins, in both their crude and purified forms, Isolate BA-90930, Streptomyces flocculus 13862 and to'rnethods for the preparation, concentration, and Isolate BA-9l124, Streptomyces flocculus 13858 isolation of these substances. These substances, strep- 3O solate IRA-9 744, Streptomyces flocculus 13856 TABLE I.-GULTURE CHARACTERISTICS OF STREPTOMYCES FLOC'CULUS, ISOLATE BA-163 Medium Amount of Growth Aerial Mycelium Sporulation Soluble Remarks Pigment Pfldh Yeast t t G t ll t 'Farcel'lp'nf Poor Nrms Spores borne in moderately long straight or wavy chains; sporophores produced singly or in pairs along hyphae; spores cylindrical, 0.65 x 1.004.130; formed by fragmentation; growth of individual colonies in three concentric circles; the other one fiat, the two central ones raised, middle one forming a narrow collar, center one mealy and velvety; rare, small spirals may have been seen on yeast agar; strong disagreeable odor. Skimed Milk Good Good;white Lacking... Pink Vege a ive mycelium cream to pink; milk not cgagulated, peptonized or hydrolyzed; no pH 0 ange. Gluwse Agar G d; hit t do do Vegetative mycelium not visible; reverse whitish; grey hit colonies similar to those on yeast extract; others with only a ring around central raised part or this may be lacking; colonies mealy, velvety.

Nutrient Agar Moderate to good.. Good; white do do Vegetative mycelium not visible; reverse white.

Synthetic Agar- Almost lacking Calcium Malate Agar Moderate to good do do .d0 Vegetative mycehum not visible; reverse white;

malate not digested.

Starch Plates Poor Poor; white do.... .do Vegetative mycelium colorless; no hydrolysis.

Gelatin Plates Moderate to good Good; white do -do Vegetative mycelium tan; reverse tan; zone of liquefaction 1.2 cm. in diameter.

Cellulose Very sparse; white growth.

Potato Plugs Moderate to goo do .do do Vegetative mycelium colorless where visible;

reverse very pale yellow.

Dextrose-Nitrate Broth Poor Sparse; white do do Vegetattive mycelium colorless; nitrates reduced to mm as.

Emerson Agar Good Good; white to -do -do yellow white.

Gluc0se-Asparagine Plates--. Moderate Good; white .do --do Vegetative mycellum tan; reverse yellowish tan; colonies rather flat except for central, somewhat irregular columnar growth.

tonigrin in particular, are useful in inhibiting both Grarn- Classification of the cultures was made by Dr. John positive and Gram-negative bacteria suiting them for a B. Routien who supplied the following description. number of applications in therapeutics, vcterinaryrnedi- The newly discovered microorganisms, X-13, BA-163 cine, industry, and agriculture. They are also useful as disand BA-472l, were identified as new strains of Strepinfecting agents, and for separating mixtures of microtomyctes flocculus (Duche). The appearance of the culture organisms for medical, diagnostic and research purposes. BA-163 which is described in Table I, after incubation The accompanying drawings illustrate characteristic 0n the media listed, agreed well with the description infrared absorption spectra of the various products of given ill y Manual of Determinafive Bacteriology, this invention, each measured in a potassium bromid pcl- 7th Edition, but certain differences between the new strain let. FIGURE I is the spectrum of streptonigrin-F, FIG- and that d i ed by I. D he in Encyclopedia Myco- URES II and III are the spectra respectively of streptonilogique 6: 1-372 (1934) were noted.

grin-A1 and A2, FIGURE IV shows the spectrum of Du-che culture grew slowly While Isolate BA-163 grew streptonigrin per se, FIGURES V and VI show respecwell, in a mass but where individual colonies were observed, they were small. Other differences included the production of a rose-colored pigment in milk while Duche described only rose-colored growth and no pigment. Isolate BA-163 failed to produce peptonization of milk in two weeks. Slow peptonization was observed by Duche. Culture BA-4721 looked almost exactly like culture BA-163 and is considered to be the same species. It was diiferent from culture BA-163 only in having numerous short, tight spirals.

Culture X-l3 showed at first a number of differences from culture BA-l63 and was thought to be either a variety of S. flocculus or perhaps a new species. Table II lists a comparison of culture X-13, as it first looked, with culture BA-163.

Cultures X-l3 and BA-163 were studied for several months with especial attention to the shape of the chains of spores of the two. After the several transfers involved over this period of time cultures X-13 and BA-i 63 came to resemble each other more and more until finally they appeared to be nearly identical.

The changes were as follows: the biochemical and physiological properties of X-13 changed to resemble those of BA-l63. Cultures BA-163 and X-13 both changed in regard to shape of the chains of spores. The straight or wavy chains of BA-163 nearly disappeared and came to be short chains in the form of curves or loops or with very small, tight spirals of one or two turns. X-13 in some areas had loops or imperfect spirals and in other areas tight spirals of one or two turns.

BA91124after seven days at 28 C. was similar in appearance to BA-907 96.

BA-35599-after seven days at 28 C. had good growth, white aerial mycelium, reverse yellowish, no soluble pigment.

BA90792-after seven days at 28 C. was similar in appearance to BA-35599 except that the reverse was yellow-brown.

BA-90926-after seven days at 28 C. was similar in appearance to BA-35599.

BA-90930-after seven days at 28 C. was similar in appearance to BA-90792.

The cultures BA-90930, BA-90926, BA-90792, BA- 35599, BA-91124, BA-90796 and BA-89818 have all now been confirmed as strains of the species Streptomyces flocculus.

In the case of Isolates X-13, BA-163 and BA-472l, the original isolates were selected on the basis of their antagonism for E. coli, and the cultures were recognized as being unlike commonly encountered antibiotic producing species. Since they had the narrow hyphae and chains of spores typical of members of the genus Streptomyces, they were planted onto media commonly used for the identification of members of this genus. Final readings of the media were made after two weeks of incubation in the accepted fashion in the case of BA-163. In the case of BA-4721, and X-13, the cultures were compared with culture BA-163 by planting each side-by-side on identification media with BA-163.

TABLE II.CULTURE CHARACTERISTICS OF STREPTOMYC'ES FLOC'CULUS ISOLA'IE X-l3 Medium Amount of Aerial Mycelium Color Soluble Remarks Growth Pigment Pridhams Yeast Extract Similar to BIL-163 except for less aerial mycelium; spores formed in abundant, long loose spirals, singly, in pairs or small whirls; spores oval, 0.6 x 1.3, formed by fragmentation.

skimmed Milk Similar to BA-163 except less growth and of a darker shade; aerial mycelium pinkish-white.

Glucose Agar Moderate...-- Moderate white. White.--- Lacking... Vegetative mycelium colorless to grayish; reverse cream to brownish, growth rather rough.

Nutrient Agar.. Similar to ISA-163 except spores white and sparse aerial mycelium,

Synthetic Agar. Similar to BA-163.

Calcium Malate Agar Sparse Sparse Whlte.. Tannish.... Vegetative mycclium cream to brownish; reverse white to tannish; malate somewhat digested.

Cellulose No growth...

Dextrose Nitrate Broth. Similar to Bit-163.

Potato Pl Similar MBA-163 except for sparse aerial mycelinrn.

Emerson Agar Moderate to Good; white to Creamy Brown.-... Vegetative mycelium colorless where visible; reverse good. pale gray. gray. brown.

Glucose Asparagine Plates Similar to ISA-163 except for dark pinkish areas of vegetative mycelium.

Starch Plates.-. Similar to Bit-163.

Gelatin Plates. Do.

Cultures BA-34885, BA-35182 and BA-35412 were compared side-by-side with BA-163 and X-13. All looked very much like BA-163 and X-l3 and showed no difference of any consequence.

It is concluded that all three cultures are representatives of the species Streptomyces flocculus to which BA-163 and X-l3 have been assigned.

The remaining cultures are described as they appeared on Emersons Agar:

BAl6986after 14 days at 28 C. had good growth, gray to light gray aerial mycelium, reverse brownishyellow and no soluble pigment. This organism has now been classified as a strain of Streptomyces fungicidicus group G, Okarni, et a1.

BA-13023--after seven days at 28 C. had good growth, white aerial mycelium, reverse yellow-brown, no soluble pigment. This organism has now been classified as a strain of Streptomyces bottropensis spiritus fabriek.

BA-89818after seven days at 28 C. was similar in appearance to BA-13023 but had a lighter yellow reverse.

BA-90744-after seven days at 28 C. had good growth, grayish-white aerial mycelium, reverse light brown and soluble pigment brown.

BA-90796aiter seven days at 28 C. was similar in appearance to BA-898l8 except that the reverse was slightly lighter in color.

It is to be understood that for the production of the Streptonigrins according to the present invention, limitation to the aforesaid organisms is not intended. It is especially desired and intended to include mutants pro duced from these strains of these organisms by various means such as irradiation with X-rays or ultraviolet light, treatment with nitrogen mustards, single cell culture techniques, and the like.

We wish also to include any organism, regardless of its appearance or physiological behavior, that may be developed by means of transformation, transduction, genetic recombination or some other genetical procedure, using a nucleic acid or an equivalent material from the herein described species, whereby it has acquired the ability to produce the elaboration product here described or to carry on the biochemical change here described.

The streptonigrins are active as antibiotic agents and streptoni grin itself is an active antiprotozoal agent in vitro. The detailed antibacterial spectrum of streptonigrin is presented in Table III. Inhibition of a number of human pathogens of clinical importance at very low concentration is observed. Some of these organisms are Corynebacteriuml diph-fheriae, Clostridium. perfringens, Salmonella typhosa, a number of strains of Proteus vulgaris, and Micrococcus pyogenes, Neisseria gonnorrhoeae, and Hemophilus influenzae. Organisms of importance in agriculture, veterinary medicine, and industry which are sensitive to streptonigrin are Listeria monocytogenes, Aerobacter aerogenes, Erwinia amylovora, Salmonella pullorum and Salmonella gallinarium, etc.

TABLE III.I-N VITRO ANTIBACTERIAL sENsI-TIVITIES TO STREPTONIGRIN Minimum inhibitory Microorganism: concentration (meg/ml.) Jl Iicrococcus pyogenes var. aureus 0.39 Streptococcus pyogenes 1.56 Erysz'pelothrix rhusiopathiae 12.5 Corynebacterium diphtheriae 0.09 Listeria monocytogenes 50 Bacillus subtilis 0.09 Clostridium perfringens 0.09 Bacterium ammoniagenes 50 Aerobacter aerogenes 3.12 Escherichia coli 0.78 Escherichia coli 21 6.25 Proteus vulgaris 6.25 Proteus vulgaris 4 3.12 Proteus vulgaris 1 6.25 Proteus vulgaris 59 6.25 Salmonella pullorum 0.78 Salmonella gallinarium 12.5 Klebsiella pneumoniae 1.56 Neisseria gonnorrhoeae 25 Hemophilus influenzae 0.19 Shigella sonnei 1.56 Erwinia amylovora 12.5 Brucella bronchiseptica 1.56 Malleomyces mallei 12.5 Desulfovibrio desulfuricans 3.12 Vibrio comma 6.25 Pasteur-ella multocida 0.19 Candida albicans 25 Saccharomyces cerevisiae 50 Micrococcus pyogenes var. aureus 376 0.78 Micrococcus pyogenes var. aureus 400 0.39 ldicrococcus pyogenes var. aureus K3 1.56 Micrococcus pyogenes var. aureus K4 0.78 Mycobacterium 607 0.19 Mycobacterium berolinense 0.19 Aerobacter aerogenes 1.56 Pseudomonas aeruginosa 6.25 Salmonella typhosa 3.12

The antimicrobial activity of the other minor components produced along with streptonigrin is given in Table IV:

TABLE IV Minimum Inhibitory Microorganism Concentration (meg/ml.)

SN A1 A2 P1 P2 Staphylococcus aureus 0.39 0. 39 0.39 50 50 Streptococcus pyogenes 0. 78 0. 39 0.78 12. 5 12. 5 Diplococcus pneumniae 0. 19 0.39 0.78 12. 12. 5 Staphylococcus aureus 376. 0. 19 0. 19 0. 19 25 25 Streptococcus faecalis 1. 56 0. 78 1. 56 25 25 Escherichia colt" 12. 5 12. 5 6. 25 100 100 Aerobacter aeroge 12. 5 12. 5 12. 5 100 100 Proteus oulgaris 3.12 12. 5 12. 5 100 100 K Zebsiella pneumoniac 3.12 12. 5 6. 25 100 100 Salmonella typhosa 6. 25 12. 5 12. 5 100 6. 25

SN-F has activity similar to streptonigrin on plates and for this reason, a tube dilution test was not run.

The SN designation used herein represents streptonigrin. SNF indicates the forecut product which precedes streptonigrin on elution from the chromatographic columns, diethylarnninoethylcellulose or silicic acid-cellulose (2:1); the two fractions which follow the major band of streptonigrin are termed SN-P and SNP (streptoni grin postcuts). The two presumably anomalous bands obtained by chromatographic separation of a crude concentrate are termed SN-A and SNA The streptonigrins are remarkably effective in treating a number of different types of infections. For this purpose, either the pure crystalline materials may be administered or one of the crude forms of the valuable products may be employed. This includes either a filtered fermentation broth prepared from Streptomyces flocculus ATCC Nos. 13257, 13535, 13536, 13850, 13851, 13852, 13855, 13857, 13858, 13859, 13860, 13861, 13862, Streptom'yces fungiciclicus ATCC 13853, Streptomyces bottropensis ATCC 13854 or a solid or liquid concentrate obtained therefrom. Such preparations should be of sufiicient potency to provide a daily dose to a lower animal host equivalent to at least about 50-500 meg/kg. of body weight. In order to do this, a concentration of the active ingredient in the carrier of at least about 0.0001% is necessary. For administration to animals, a nontoxic carrier of course, is selected. By non-toxic is meant a carrier which is non-toxic when administered in a quantity sufiicient to provide the above dose of the particular streptonigrin. This may be either a pharmaceutical carrier, either a liquid or a solid such as water, aqueous ethanol, syrup, isotonic saline or glucose, starch, lactose, calcium phosphate, etc., an animal feed stuff, or a mixture of various materials as occurs in a filtered fermentation broth. Either oral or parenteral administration is satisfactory. The parenteral route is perhaps preferred until a satisfactory regimen adapted to the patient is developed.

The present invention embraces the process for growing Streptomyces flocculus ATCC Nos. 13257, 13535, 13536, 13850, 13851,13852, 13855, 13857, 13858, 13859, 13860, 13361, 13862, Streptomyces fungicidicus ATCC 13853, Streptomyces bottropensis ATCC 13854 and Isolate BA-90744 under controlled conditions to produce the streptonigrins. Biologically active filtrates are satisfactorily prepared, for instance, using an aqueous nutrient medium containing 10 g./l. of glucose, 15 g./l. soybean meal, 5.0 g./l. corn meal, 2.5 g./l. distillers solubles, and 2.0 g./l. of calcium carbonate. This broth is adjusted to pH 7.0 before autoclaving. Inocula may be either spore suspensions or preformed growth of the organism. Fermentation is carried out at 28 C. either by shake culture or small pot fermentations, mechanically aerated, for from about 60 to hours. Progress of the fermentation is followed by standard plate assay methods using either B. subtilis, Staph. aureus, No. 376, or E. coli No. 21 as the assay organism.

A wide variety of fermentation media have been tested and found to be satisfactory. A medium composed basically of a source of nitrogen, of a carbohydrate, and of minerals is required. Satisfactory nitrogen sources include myriad proteinaceous materials such as hydrolyzed casein of various types, soybean meal, casein, distillers solubles, corn meal, etc. Suitable carbohydrate sources are dextrose, glycerol, dextrin, starch, etc. Less satisfactory results are obtained on lactose-corn steep mixtures. The preceding materials frequently contain sufiicient minerals to satisfy the mineral requirements of the organism without the addition of substantial additional mineral components.

After a satisfactory level of antibiotic activity is attained, following a 6080 hour fermentation, the beer is filtered employing from 35% by weight of a diatomaceous filter aid. The antibiotic content of the filtered broth may be extracted therefrom with a Water immiscible organic solvent such as preferably n-butanol, methyl isob'utyl ketone, or ethyl acetate, or somewhat less satisfactorily with ether, benzene, toluene, methylene chloride, chloroform, or carbon tetrachloride. Approximately 30% of the total antibiotic activity remains in the mycelial cake when beer filtration is carried out at pH 4.0. This portion is readily recovered by extraction of the cake with a lower alkanol such as methanol, ethanol, or isopropanol.

It is preferred, when employing solvent extraction for the recovery of the streptonigrins from fermentation broths, to first adjust the broths to a neutral or slightly acidic pH. The range of from about pH 3.5 to 7.2 is prefered. Extraction is relatively more rapid and complete at pH 3.5 than at pH 7.2.

As indicated previously, more than one antibiotically active substance is produced during fermentation of Streptomyces flocculus ATCC Nos. 13257, 13535, 13536, 13850,13851,13852, 13855, 13857, 13858, 13859, 13860, 13861, 13862, Streptomyces fungfcidious ATCC 13853, Streptomyces bottropensis ATCC 13854 and Isolate BA-90744. When ethyl acetate or n-butanol is used as the extracting solvent, the extract is clarified and concentrated under reduced pressure to 1/50 volume. The mycelial cake is extracted with a 1:4 mixture of 0.3% phosphate buffer (pH 4.0) and ethyl acetate for several hours. The extract is concentrated and added to the concentrate from the broth. The combined concentrate is treated with four to five times its volume of ligroin or isopropyl ether, the bulk of the active material is extracted from the organic solvent mixture by shaking with successive portions of a phosphate buffer (pH 7.5) until a fresh portion of buffer extract turns only a light brown. A significant amount of activity, however, remains in the organic layer. The substance responsible for this activity appears to be a different but closely related antibiotic which is somewhat less acidic in character than streptonigun.

The aqueous layer is extracted with /2 volume of isopropylether and the organic phase discarded. The aqueous phase is adjusted to pH 4.0 and extracted with chloroform. On concentration of the chloroform phase, crystals of the streptonigrin complex (I), 80-90% purity, are obtained.

The various components are separated by column chromatography on diethylaminoethylcellulose or silicic acidcellulose or by countercurrent distribution in the system ethyl acetate-3% phosphate buifer, pH 7.5. The products are recrystallized from pyridine-ethanol solution or from acetone-ethylacetate: (1:4 in each instance). The pure products are brown or red-brown in color.

When using a diethylaminoethylcellulose column -20 g. per gram of sample), the crystalline sample, (I) dissolved in a 1:3 pyridinezmethanol solution, is added to the column and developed first with methanol and then with methanol containing increasing amounts of 1% acetic acid. A minor component (SN-F) appears first followed by the major component, streptonigrin (SN), and then two additional minor components (SN-P and SN-P Alternatively, and preferably, a column prepared from a 2:1 mixture of silicic acid and cellulose in chloroform is used, and a chloroform solution of the sample (I) run through the column which is developed with chloroform. Here also, a minor component precedes the major streptonigrin component. Further washing of the column with chloroform containing increasing amounts of ethyl acetate produces at least two additional minor components. However, when product I is obtained in non-crystalline form and run through a silicic acid-cellulose column as described above, two anomalous streptonigrin-like components (SN-A and SN-A are obtained. These components appear almost identical with respect to ultraviolet extinction (Ej g values) infrared spectra, melting point and microbiological activity. A mixture of the two components with streptonigrin fails to separate on passage through another silicic acidcellulose column. It appears that the separation may have been due to some carrier substance in the crude concentrate.

An alternative work-up procedure consists in filtering the broth, adjusting to pH 4.0 and stirring the filtrate with 12% (by volume of fullers earth) for 30 minutes. The slurry is filtered with the aid of Supercel (a diatomaceous earth filter aid) the cake washed with water and extracted twice with methanol, acetone or ethyl acetate. The combined extracts are concentrated to small volume and processed as described above. The mycelial cake is extracted separately as described above. The combined extracts, more correctly the concentrates of the combined extracts, are processed together.

Streptonigrin, a complex structure comprising three ring systems: quinonoid, pyridine and benzenoid, has the formula:

Its systematic name is 5-amino-6-(6-methoxy-5,8-dihydro-7-amino-5,8-dioxo 2 quinolyl) 4 (2 hydroxy- 3,4-dimethoxyphenyl)-3-methylpicolinic acid. It is an acidic substance which behaves as a weak monobasic acid. Potentiometric titration in dioxane above shows an apparent pK value of 6.3-6.5. Its composition, determined by averaging the values of several microanalyses on crystalline streptonigrin, is 59.11% C; 4.48% H; 10.69% N; 18.35% methoxyl 25.72% oxygen (by difference). Group analyses indicate three methoxyl groups and one C-methyl group. These data, when considered in conjunction with studies on derivatives of streptonigrin including degradation products, especially nuclear magnetic resonance and mass spectral data, show the molecular formula to be C l-I O N Pure crystalline streptonigrin is dark maroon to black in color and crystallizes from acetone-ethyl acetate (1:5) as thin elongated rhombohedral plates. Individual crystals of the substance appear to be orange when viewed through a microscope using transmitted light. The crystalline form and, to some extent, the color depend on the nature of the crystallizing solvent. For example, chloroform and ethyl acetate produce brown rectangular plates; aqueous acetone produces long, pale brown needles, and from tetrahydrofuran bright reddish brown rectangular plates separate. On slow heating, streptonigrin begins to darken at 268 C. and melts with decomposition at 272-275 C. On rapid heating it darkens at 272 C. and melts at 275-277 C. with decomposition. It exhibits absorption maxima at the following wave lengths in the infrared region of the spectrum when measured in a KBr pellet containing 1% of the product: 3448, 3400, 3356, 3300, 3268, 2900, 2800, 1739, 1675, 1631, 1605, 1580, 1560, 1500, 1460, 1432, 1425, 1400, 1370, 1334, 1285, 1225, 1196, 1170, 1094, 1084, 1033, 1000, 956, 916, 866, 821, 810, 778, 748 and 704 cmr The curve is more particularly illustrated in FIGURE IV. When the pure crystalline substance is dissolved in methanol at a concentration of 1%, absorption maxima in the ultraviolet region of the spectrum appear at 245 mu, E=45,000; 375 mu, E=19,500.

Streptonigrin is only slightly soluble in water at neutral and acidic pHs. Its acidic character is reflected by its relatively greater solubility in aqueous sodium bicarbonate or in buffers of pH 7.0 or higher. In addition, the substance appears to be unstable at alkaline pH values. In aqueous sodium hydroxide, for example, it dissolves to form a deep greenish yellow solution which changes to brown or red-brown with evolution of ammonia. It is very slightly soluble in ether, benzene, and methylene chloride, and moderately soluble in ethyl acetate, the lower alkanols from methanol to butanol, and acetone. It dissolves in concentrated sulfuric acid to give a bright yellow solution. Alcoholic ferric chloride test solution turns a dark greenish-brown color when streptonigrin is dissolved in it. Its reddish brown aqueous solution changes to a bright yellow color when treated with sodium bisulfite. When this yellow bisulfite solution is shaken with water immiscible solvents in which streptonigrin is soluble, such as ethylacetate or chloroform, the extract becomes reddish-brown and contains an antibiotically active solute. These properties and reactions are attributed to the quinone type structure. Since streptonigrin is an acidic substance, it forms salts with basic substances such as metal hydroxides, basic metal salts, ammonia, hydrazine, amines such as mono-, diand trialkylamines, alkanolamines, ethylenediamine and its N-substituted derivatives, etc. The streptonigrin salts of the alkali and alkaline earth metals such as sodium, potassium, lithium, calcium, magnesium, etc., are convenient dosage forms due to their solubility. The acid character is further borne out by the esterification and amidation (including with hydrazine) of streptonigrin. Treatment with a slight excess of diazomethane in ether or with methyl alcohol and an acid catalyst, such as mineral acids, the hydrogen form of Dowex 50 (an ion exchange resin which is a copolymer of sulfonated polystyrene and divinylbenzene) or aromatic sulfonic acids, gives the monomethyl ester. Its composition, determined by microanalysis, is C, 59.88%; H, 4.75%; N, 10.51%; 0, 24.86% and methoxyl 23.77% in agreement with the formula C H O N Reaction with dimethylsulfate produces the same compound. Higher alkyl esters are prepared by substitution of the appropriate alcohol for methyl alcohol. Further methylation of the monomethylester with dimethylsulfate produces dimethyl streptonigrin, an ether ester, having the formula C I-1 19 Saponification of the dimethyl derivative with sodium hydrosulfite gives a monomethyl compound, an ether, which is isomeric with the methyl ester. The product is weakly acidic and forms a dark greenish brown color with ferric chloride. These methylation reactions thus indicate the presence of a phenolic group and a possible carboxyl group in streptonigrin. The remaining components produced with streptonigrin likewise form esters and salts; and exhibit the same solubility characteristics and color reactions as does streptonigrin. The red-brown chloroform and ethyl acetate extracts of their bisulfite treated solutions are antibiotically active.

The methyl ester of streptonigrin, a neutral, dark brown crystalline compound; M.P. 270275 C. (dec.), is only slightly soluble in water and the common organic solvents such as lower alcohols, ethyl acetate and acetone and insoluble in aqueous sodium bicarbonate. It does not produce a color with alcoholic ferric chloride. Its infrared spectrum, measured in a KBr pellet containing 1% of the product (FIGURE VII), bears a strong resemblance to that of streptonigrin. Absorptionmaxima occur at 3472, 3322, 2933, 2841, 1715, 1689, 1610, 1595, 1555, 1513, 1471, 1443, 1410, 1383, 1351, 1311, 1294, 1230, 1205, 1116, 1089, 1076, 915, 868, 801, 754 reciprocal centimeters. Its ultraviolet spectrum is very similar to that of streptonigrin: absorption maxima at 245 and 375 mu with =E=43,000 and 19,500 respectively.

Amide or hydrazide formation at the carboxy group according to standard methods produces derivatives. Prolonged treatment (2 days) of a dioxane solution of the methyl ester of streptonigrin with hydrazine hydrate at room temperature produces streptonigrin hydrazide. This product crystallizes from chloroform-methanol (1-1) as a dark brown, crystalline solid; M.P. 275800 C. (dec.).

Further evidence for a quinonoid structure is based upon the ready reduction of streptonigrin by sodium dithionite, lithium aluminum hydride or hydrogen in the presence of palladium. In each case a yellow, unstable dihydro derivative which is readily reoxidized to streptonigrin by air is obtained. Reductive acetylation with acetic anhydride, sodium acetate, platinum and hydrogen yields a yellow crystalline solid which analyzes for C H O N in agreement with a triacetyl derivative of dihydrostreptonigrin. The product, an acidic substance, pK,,=4.3-4.5, is soluble in aqueous sodium bicarbonate and gives a positive ferric chloride test.

Gentle treatment of streptonigrin with aqueous alkali produces desaminostreptonigrin, a brick-red crystalline solid apparently formed by exchange of an amino group by a hydroxy group. This product melts at 270 275 C. (dec.) and contains the elements carbon, hydrogen, nitrogen and oxygen in the following proportions by weight: C, 58.0; H, 4.30; N, 8.0; O, 29.7; (by difference); methoxyl, 17.70; CCH 2.75; (C H O N It is a dibasic acid with pK =4.8 and 6.7 and an equivalent weight of 250-260. Methylation with methanolic acid yields a monomethyl derivative (C H O N while diazomethane gives a dirnethyl derivative (C27H25O9N3) which is neutral and gives no ferric chloride reaction.

Acetylation of streptonigrin produces a variety of products depending upon the conditions used. Acetylation at room temperature with acetic anhydride and pyridine produces at least two isomeric monoacetylated derivatives of formula C H O N Acetate I, an O-acetyl derivative, is obtained as deep brown rectangular plates on crystallization from pyridine-ethanol; M.P. 1202l2 C. It is weakly acidic (pK,,=6.1-6.3 in 50% aqueous dioxan) and its infrared spectrum is very similar to that of streptonigrin, Acetate II, an N-acetyl derivative is obtained as brownish-pink needles from pyridine-ethanol; M.P. 237- 239 C. It is a stronger acid (pK,,=4.3-4.5) than the O-acetyl isomer. The O-acetyl derivative is considerably more active as an antitumor agent than the N-isomer. Acetylation with acetic anhydride and an acid catalyst, such as boron trifluoride, sulfuric or perchloric acid produces a yellow, crystalline new quinonoid tetraacetyl derivative; M.P. 206208 C. having the formula aeration of the contents with up to two or more volumes of air per minute. A satisfactory medium for large scale production is given above. The growth of the microorganism and antibiotic production usually reaches its maximum, as measured by one of the above assay methods, after about 60 to hours at 28 C. However, variation in the equipment used, the rate of aeration, stirring, and so forth frequently affect the time period for maximum activity to be reached. A period of at least about 24 hours is required in any case. Aeration of the medium on submerged growth is maintained at the rate of about /2 to 2 volumes of air per volume of broth per minute. Aseptic conditions, of course, must be maintained throughout the transfer of the inooulum, and the growth of the microorganism. Removal of the mycelium and recovery of the antibiotic is carried out as described above.

streptonigrin and its alkyl esters are somewhat toxic materials. However, therapeutic doses can be administered without substantial adverse elfect. This is illustrated by the data in Table VII. Swiss white mice where given five consecutive daily injections of 1 cc. aqueous solutions or suspensions containing the dosages of streptonigrin and its methyl ester, respectively, listed, and survival rates noted.

TABLE V.-TOXICITY OF STREPTONIGRIN AND ITS METHYL ESTER The following examples are helpful to further illustrate the invention in detail. They are not, however, the sole embodiments, and are not considered to limit the scope thereof in any way.

EXAMPLE I A nutrient medium having the following composition is prepared, adjusted to pH 7.0, and sterilized: Glucose l g./l.; Soybean meal 15 g./l.; distillers solubles 2.5 g./l.; dipotassium phosphate 5.0 g./l.; sodium chloride 2.0 g./l.; calcium carbonate 2.0 g./l.; tapwater to volume. Inoculum is prepared by transferring the growth of a well sporulated slant of Streptomyces flocculus Isolate BA-163 to a portion of this medium and incubating for about 36 to 40 hours at 28 C. on a rotary shaker. The main batch of medium is then inoculated by mixing With by volume of the inoculum so prepared. Aeration is effected at a rate of about 2 volumes of air per minute and efiicient agitation is provided during incubation at 2630 C. Progress of the fermentation is followed by plate assay of the filtered broth at intervals employing Staph. aureus No. 376 and E. coli No. 21 as the assay organisms. Typical zones of inhibition of harvestable broth are 27.0 mm. and 21.0 mm. respectively.

EXAMPLE II A fermentation broth is prepared as described in Example and clarified by filtration employing from about 3 to 5% by weight of a diatomaceous earth filter-aid. The filtrate is then adjusted to pH 4.0 and extracted with /3 volume of ethyl acetate. The ethyl acetate extract is then filtered to free it of insoluble extraneous matter, and concentrated, at 25-30 C. under reduced pressure, to a small volume. Approximately 30% of the antibiotic activity produced remains in the mycelial cake. This is recovered by stirring the wet-cake with methanol, filtering, and concentrating the aqueous methanol extract until most of the methanol has evaporated therefrom. This concentrate is then adjusted to pH 4.0 and extracted with approximately an equal volume of ethyl acetate. The two ethyl acetate concentrates are then combined and treated with five volumes of isopropyl ether. This solution is then shaken several times with a pH 7.5 phosphate butter until the last extract is only a light brown in color. The combined aqueous buffer extract is then adjusted to pH 4.0 and extracted several times with methylene chloride. Concentration of the methylene chloride extract and cautious treatment of the concentrate with ether results in the precipitation of impure streptonigrin as thin elongated rhombohedral plates, darlcmaroon to black in color.

Column chromatography of this material according to the procedure of Example 111 brings about separation and purification of streptonigrin and the fractions SN-F, SN- P1 and SN-Pg.

EXAMPLE III A fermentation broth is prepared as in Example 1 using S. flocculus, Isolate Elk-34885 in place of S. flocculus, Isolate BA-163. The beer is filtered, the broth adjusted to pH 4.0 and extracted with /2 volume of n-butanol. The extract is clarified and concentrated to 3 of the original volume. The mycelial cake is extracted with 1:4 mixture of 3% phosphate buffer, pH 4.0, and ethyl ace- 123 tate for several hours. The extract is concentrated and added to the concentrate from the broth.

The concentrate is processed further as shown in the following scheme.

Flow Sheet for the Recovery of Streptontgrln Complex Concentrate Stir with 1-2 volumes of 2-37. Phosphate buffer, pH 7.5, and o volumes of llgroint Check and adjust pH of the aqueous layer to 7,5. Separate the aqueous layer and repeat twice more with buffer.

Lig roin layer Extract once with a 3:1. mixture of Aqu'eous layer Stir with 1/2 volume of isopropyl ether and separate Iscpropyl Aqueous layer Aqueous Ligroin other layer methanol. layer (discar Adjust to pH 1;.0 layer Lenard) and extract 1V twice with chloroform chloroform layer Aqueous layer concentrate Extract once The crystals from (1) contain the bulk of the active material from the concentrate. The product is -90% pure as streptonigrin. The mother liquors from the crystals are added to IV and processed further. Likewise, I1 and III are combined and worked up together.

The various components are separated and purified by chromatography on a column prepared from a 2:1 mixture of silicic acid and cellulose in chloroform. The crystalline sample (-I) is dissolved in chloroform, added to the column and developed with more of the same solvent. After the main band is eluted, the column is washed with chloroform containing increasing amounts of ethyl acetate: 10, 25 and 50%. The following activity distribution is observed. A minor band l2% (SN-F), major band 80% (streptonigrin), two minor bands 3- 5% each (SN-P SN-P The properties of the various components are summarized below:

Ultraviolet Spectra I Compound 7 Max. E 7 Max. 13

1 cm. 1 cm 850 375-380 my... 370 825 375-380 n1,u 350 700 375-380 mg. 340 800 375-380 mun" 356 Meltingcloints, Analysis o H N -187 (dec.) 60. 54 4. 36 11. 42 272-275 (dec.) 59. 11 4. 48 10. 69 -197 (doc.)- 57.97 4. 72 11.02 SN-Pz 187-195 (dec.) 58. 12 4. 50 10. 60

The product, actually obtained as a hemihydrate (O, 58.20%; H, 4.56%;

N, 10.79%), is dried at 150 C. in vacuo to give the anhydrous form.

The various components are readily purified by recrystallization from pyridine-ethanol solution.

The infrared spectra of the components when measured in KBr pellets containing 0.3% of the product exhibit absorption maxima at (in reciprocal centimeters):

SN-F (FIG. I).3410, 3330, 3250, 2920, 1735, 1680, 1630, 1600, 1584, 1564, 1478, 1440, 1400, 1370, 1339, 12.70, 1230, 1208, 1180, 1162, 1100, 1092, 1071, 1050, 1036, 1008, 918, 873, 860, 828, 822, 806, 788, 760, 744, 702, 684, 658.

SN (FIG. IV).3448, 3400, 3356, 3300, 3268, 2900, 2800, 1739, 1675, 1631, 1605, 1580, 1560, 1500, 1460, 1432, 1425, 1400, 1370, 1334, 1285, 1225, 1196, 1170, 1094, 1084, 1033, 1000, 956, 916, 866, 821, 810, 778, 748, 704.

SN-P (FIG. V).3410, 3340, 3220, 2910, 2820, 1719, 1680, 1610, 1588, 1550, 1500, 1461, 1440, 1405, 1372, 1344, 1318, 1280, 1230, 1186, 1091, 1072, 1047, 1023, 995, 919, 890, 868, 814, 804, 784, 746, 710, 682, 652.

SN-P (FIG. VI).3320, 3210, 2900, 1715, 1675, 1630, 1608, 1570, 1555, 1500, 1460, 1440, 1405, 1375, 1342, 1280, 1228, 1184, 1090, 1072, 1045, 1022, 995, 918, 890, 868, 814, 782, 747, 709, 682.

Fraction 1V and the combined fractions II and III, which are richer in the minor components, are worked up in like manner to give additional quantities of the various components.

EXAMPLE IV The procedure of Example III is repeated. However, the fraction I, which contains some oily material (apparently not removed by extraction), is not obtained in crystalline form and, on running through the silicic acidcellulose column, produces five bands having the following distribution:

Band Percent 14 C.). The aqueous layer is separated and the solvent layer extracted twice more with isopropyl ether to remove adhering oily impurities and separated. The buffer layer 15 then acidified to pH 4.0 and extracted twice with /2 volume of chloroform. The aqueous residue is finally extracted once with /2 volume of ethyl acetate.

The mycelial cake is extracted twice with sufficient ethyl acetate to give a thin slurry in the presence of /5 volume (based on the ethyl acetate) of a 5% solution of phosphate buffer, pH 4.0. The two solvent extracts are combined, concentrated to a small volume and added to the chloroform extract from the broth. The mycelial extract provides about 20-30% more of the active material.

The various components are separated by chromatography on a column of silicic acid and cellulose powder. A mixture of 2 parts of silicic acid (Mallinkrodt, 100 mesh) and one part of cellulose powder (Solcafloc, Brown & Co., Berlin, NH.) is made into a slurry with chloroform and poured into a column. About 25 g. of this mixture is suitable per gram of the substance. The sample, dissolved in chloroform is added to the column which is then developed with chloroform. The bands which move with this solvent are eluted off the column. The solvent is progressively changed to 10, 25 and ethyl acetate in chloroform. The products are recovered by evaporation.

In general the elution pattern of the components is as follows: A minor component the most solvent-soluble, is obtained just behind the solvent front. Then comes the major component streptonigrin, which accounts for almost 75% of the total mixture. Next there are at least two minor components eluted chiefly when 10% ethyl acetate in chloroform is used. In order to keep the name of streptonigrin unchanged for the major component the following designation is adopted for the minor components. The component that appears before the major band is designated by the letter F (for fore cut); and the fractions that follow the major band by the letter P are more polar. 40 (P P for post cuts).

UV Spectra Analysis Component 7 max. M.P. C.) C H H 'SN-A {gggggfi 2%? }268275 (dec.) 58'69 11'00 SN-A 330 }270-275 (dee.). 59-12 1955 sN my }272-215 (dec.) 58-20 1979 EXAMPLE V A fermentation broth prepared as described in EX- ample I is filtered, adjusted to pH 4.0 and extracted once with /2 volume on n-butanol. The extract is concentrated azeotropically under reduced pressure (35 C.) to remove most of the butanol. The concentrate is shaken with ethyl acetate twice to transfer all the active material into the organic solvent. This extract is concentrated to small volume and stirred with 2 volumes of 3% phosphate buffer, pH 7.6 To this vigorously stirred mixture is added 5l0 volumes of ligroin (13.1. 60-90 EXAMPLE VI The streptonigrin complex (I) produced as described in Example III, is purified by passage through a column of diethylaminoethylcellulose in methanol (15 g. per gram of sample). The sample (1) dissolved in pyridinemethanol (1:3) is added to the column and development carried out with 0.1% acetic acid in methanol. The order of bands observed here is the same as that observed in Example III using a silicic acid-cellulose column.

EXAMPLE VII Repetition of the procedures of Examples I and II using the following microorganisms in place of S. Flocculus, Isolate BA-163, produces streptonigrin together with the minor components previously described:

Isolate: ATCC BA89818Strept0myces flocculus 13855 BA90744Streptomyces flOccltlMs 13856 BA-90792-Streplomyces flocculus 13860 BA90796Strept0myces flocculus 13857 BA90926Streptomyces flocculus 13861 BA-90930-Streptomyces flocculus 13862 BA-9l124-Szrept0myces flocqulus 13858 15 EXAMPLE VIII A fermentation broth is prepared as described in Example I, and aseptically filtered through a bacteriological filter to provide a sterile filtrate which is frozen and dried from the frozen state. The dried residue then is a composition containing about 0.1% by weight of streptonigrin.

EXAMPLE IX Following the procedure of Example II, but using A 0.0001% solution of pure crystaline streptonigrin, prepared as described in Example II, is aseptically prepared using isotonic saline as the vehicle. This solution is then satisfactory for parenteral use in the treatment of sensitive infections.

EXAMPLE XII Equivalent quantities of pure crystalline streptonigrin (Example II) and of potassium hydroxide are dissolved in water, filtered, and the clear solution frozen and dried from the frozen state. The powder remaining is the potassium salt of streptonigrin.

In similar fashion, calcium hydroxide, magnesium hydroxide, sodium hydroxide, and lithium hydroxide yield the calcium, magnesium, sodium, and lithium salts.

The metal salts of SN-F, SN-P SN-P SN-A and SN-A are likewise prepared by substituting the desired component for streptonigrin.

EXAMPLE XIII Equivalent quantities of sodium acetate and streptonigrin are dissolved in glacial acetic acid. The mixture is frozen, and dried from the frozen state to afford the sodium salt of streptonigrin.

*EXAMPLE XIV Methyl ester of streptonigrin METHOD A.DIAZOMETHA'NE To a solution of streptonigrin in dioxane there is added a slight excess of diazomethane in ether solution. After standing at room temperature for approximately 5 min., the solution is evaporated to dryness. The residue is taken up in methylene chloride and washed once with 2% aqueous sodium bicarbonate to remove unchanged streptonigrin. The methylene chloride layer is washed with Water, concentrated to small volume and an equal volume of ether added. The methyl ester crystallizes as grey-brown rectangular plates; M.P. 270-275 C.

METHOD B.\IETHANOL A mixture of streptonigrin (1 gm.), methyl alcohol (25 ml.) and Dowex50, hydrogen form, (1 gm.) are refluxed gently for about 2 hours. The reaction is checked for completion by treating an aliquot of the solution with ferric chloride; the absence of color being indicative of complete reaction. The mixture is then filtered and the filtrate evaporated to dryness to give the methyl ester. An additional quantity of product is obtained by extracting the resin with pyridine (30 1111.), concentrating the extract and precipitating the methyl ester by addition of methyl alcohol.

The same product is obtained by each method. It is a grey-brown crystalline material; M.P. 270275 C.

Analysis.Calcd. for C H O N 59.99% C; 4.65% H; 10.77% N; 24.59% 0 (by difference); 23.80% methoxyl. Found: 59.88% C; 4.75% H; 10.51% N; 24.86% 0 (by difference); 23.77% methoxyl.

Higher alkyl esters are likewise prepared by substitution of methyl alcohol by ethyl, propyl and butyl alcohols.

In like manner, components SN-F, SNP SN-Pg, SN- A and SN-A are transformed to their respective esters.

EXAMPLE XV Dimethyl streptonigrin Dirnethyl sulfate (2 cc.) is added to a solution of streptonigrin (1 g.) in acetone cc.). Potassium carbonate (10 g.) is added, the mixture refluxed for 6 hours, filtered and concentrated to small volume. The product which separates is crystallized from chloroform-methanol (1-1); M.P. 230232 C.

Analysis.Calcd. for C2'7H26O3N4: 60.67% C; 4.90% H; 10.48% N; 23.95% 0 (by difference); 29.03% methoxyl. Found: 60.74% C; 4.95% H; 10.71% N; 23.60% 0 (by difference); 28.39% methoxyl.

Application of this procedure to the methyl ester of streptonigrin produces the same product.

EXAMPLE XVI Methyl ether of streptonigrin The dimethyl streptonigrin product of the preceding example (1 g.) is dissolved in 100 cc. of methanol. The solution is heated to reflux, nitrogen has bubbled through the solution, and a solution of 1 g. sodium hydrosulfite in 5 cc. water added. (The solid becomes a bright yellow on addition of the hydrosulfite.) Potassium hydroxide (5 g. in 10 cc. water) is added and the mixture refluxed for one hour. Glacial acetic acid (10 cc.) is added, the flow of nitrogen discontinued, and the mixture cooled, concentrated and shaken between chloroform-water. The chloroform extract is dried over anhydrous sodium sulfate, concentrated and the product crystallized from chloroform-methanol (1-1), M.P. 270 C.

Analysis.Calcd. for C H O N 59.99% C; 4.65% H; 10.77% N; 24.59% 0 (by difference); 23.80% methoxyl. Found: 59.68% C; 4.82% H; 11.03% N; 24.59% 0 (by difference); 23.29% methoxyl.

EXAMPLE XVII Acetylation of streptonigrin A. MONOACETYLATION streptonigrin (1 g.) in acetic anhydride (10 ml.) and pyridine (2 ml.) is held for 2-3 days at room temperature. The solution is then poured into water, acidified with dilute sulfuric acid and extracted with chloroform. The extract is concentrated and put on a chromatographic column of silicic acid in chloroform. Elution first with chloroform followed by 1:3 ethyl acetate-chloroform gives two major bands. The corresponding fractions from each band are concentrated and the products crystallized from pyridine-ethanol.

Acetate (I) is obtained from the first band. It crystallizes as deep brown rectangular plates; M.P. 2102l2 C.

Analysis.-Calcd. for C H O N C, 58.90; H, 4.76; N, 10.18; acetyl (I), 7.82. Found: C, 58.42; H, 4.46; N, 9.68; acetyl, 7.63.

Acetate (II) is obtained from the second hand. It crystallizes as brownish pink needles; M.P. 237-239 C.

Analysis.Calcd. for C H O N C, 58.90; H, 4.76; N, 10.18; acetyl (I), 7.82. Found: C, 57.89; H, 4.51; N, 10.02; acetyl, 7.65.

B. TETRA-ACETYL DERIVATIVE A solution of streptonigrin in acetic anhydride 10 ml.) and boron trifluoride-etherate (2-3 drops) is stored for acetyl, 19.27.

' 24 hours at room temperature. The solution is then poured 1 into water, the solid filtered and crystallized from acetoneethanol. The acetate separates as bright yellow rectangular prisms, M.P. 206208 C. Yield, 0.7 g.

Analysis.Calcd. for C H O N C, 57.22; H, 4.66; N, 8.09; acetyl (4), 24.86. Found: C, 56.42; H, 4.96; N, 8.03; acetyl, 24.89.

EXAMPLE XVIII Desaminostreptonigrin EXAMPLE XIX Methylation' of desaminostreptonigrin A. MONOMETHYL DESAMINOSTREPTONIGRIN Desaminostreptonigrin (1 g.) is refluxed in methanol (50 ml.) with boron trifluoride-etherate (1 ml.) for 20-24 hours. The crystalline solid which separates is filtered and recrystallized from pyridine-methanol. The methyl ester separates out as coppery brown plates; M.P. 240242 C. Yield, 0.6 g.

Analysis.Ca1cd. for C H O N H O: C, 57.88; H, 4.67; N, 7.79; OCH 23.80. Found: C, 58.44; H, 4.60; N, 7.95; OCH 22.52.

B. DIMETHYL DESAMIN-OSTREPTONIGRIN Desaminostreptonigrin (1 g.) is dissolved in dioxane (25 ml.) and treated with an excess of diazomethane. After 5 minutes, acetic acid is added to decompose the diazomethane and the solution concentrated. The concentrate is shaken between chloroform and aqueous bicarbonate and the solvent layer separated and concentrated to dryness. The produce is crystallized from a mixture of chloroform and methanol. The dimethyl derivative forms brick-red rectangular plates; M.P. 225-228 C. Yield, 0.7 g.

Analysis.Calcd. for C H O N C, 60.56; H, 4.71; N, 7.85; OCH 28.97. Found: C, 59.89; H, 4.93; N, 7.64; OCH 28.75.

EXAMPLE XX Triacetyl dihydrostreptonigrin Streptoni-grin l g.) is hydrogenated in a mixture of acetic anhydride (5 ml.), anhydrous sodium acetate (1 g.) and Adams catalyst (0.2 g.) in a Parr apparatus for 24 hours. The mixture is filtered and diluted with water. After 15 minutes the precipitated solid is filtered and washed with water. It is taken up in chloroform and shaken once with cold aqueous sodium bicarbonate. The aqueous layer is acidified, the solid filtered and crystallized from ethanol. Triacetyl dihydrostreptonigrin separates as pale yellow needles;yield 0.5 g.

Analysis.-Calcd. for C H O N C, 58.67; H, 4.77; N, 8.83; acetyl, 20.35. Found: C, 58.26; H, 4.83; N, 8.74;

EXAMPLE XXI streptonigrin hydrazide To a solution of 0.5 g. of streptonigrin methyl ester in 2 cc. of dioxane is added 2 cc. of hydrazine hydrate (99% The mixture is allowed to stand at room temperature for 2 days, then poured into water. The aqueous mixture is extracted with choloform, the chloroform extract dried with anhydrous sodium sulfate and evaporated to dryness. The product is crystallized from 1:1 chloroformzmethanol as dark brown crystals; M.P. 275280 C. (dec.).

18 In like manner substitution of hydrazine by ammonia produces the amide of streptonigrin.

EXAMPLE XXII Ammonium salt of streptonigrin taining product which comprises cultivating a strain of microorganism selected from the group consisting of ATCC S treptomyces flocculus 13 25 7 Streptomyces floccwlus l 35 3 5 Streptomyces floc'cul us 135 36 Streptomyces bottropensz's 13854 Streptomwces fungicidus 1385 3 Streptomyces flocculus 13 852 Streptomyces flocculus 13 1 S treptomyces flocculus 1 3 850 S treptomyces flocculus 13 859 Streptomyces flocculus 13 855 Isolate BA90744 13 856 Streptomyces flocculus 13 860 Streptomyces flocculus 13 857 Streptomyces flOlCCMlMS 13 861 Streptomyces flocculus 13862 Streptomyces flocculus 13 85 8 in an aqueous nutrient medium under submerged aerobic conditions until substantial'antibiotic activity is imparted to said medium.

2. A process as in claim 1 wherein the antibiotic substances are recovered from the fermentation broth.

3. The process of claim 2 wherein the antibiotic substances are recovered by solvent extraction.

4. The process of claim 2 wherein the antibiotic substances are recovered by adjusting the fermentation broth to a pH of up to about pH 7.2, filtering the mycelium and extracting the filtrate with a water immiscible organic solvent.

5. The process of separating a mixture of antibiotic substances streptonigrin F, streptonigrin A streptonigrin A streptonigrin, streptonigrin P and streptonigrin P which comprises contacting a chloroform solution thereof with silicic acid-cellulose (2:1) whereby the antibiotic substances are adsorbed, selectively eluting the antibiotic substances therefrom by means of chloroform containing increasing amounts of ethyl acetate and recovering said antibiotics therefrom.

6. The process of separating a mixture of antibiotic substances streptonigrin F, streptonigrin A streptonigrin A streptonigrin, streptonigrin P and streptonigrin P which comprises contacting a pyridinemethanol (1:3) solution thereof with diethylaminoethylcellulose whereby the antibiotic substances are adsorbed, selectively eluting the antibiotic substances therefrom with methanol containing increasing amounts of 0.1% acetic acid and recovering said antibiotics therefrom.

7. The process of separating a mixture of antibiotic substances streptonigrin F, streptonigrin A streptonigrin A streptonigrin, streptonigrin P and streptonigrin P; which comprises countercurrent distribution in the system ethylacetate3% phosphate buffer (pH 7.5) and recovery of the said antibiotics therefrom.

8. A compound selected from the group consisting of 5 amino 6 (6 methoxy '4 5,8 dihydro 7 amino- 19 5,8 dioxo 2 quinolyl) 4 (.2 hydroxy 3,4 dimethoxyphenyl)-3-methylpicolinic acid, the lower alkyl esters, the monomethyl ether, the hydrazide, the ammonium and the alkali metal and alkaline earth metal salts thereof.

9. 5 amino 6 (6 methoxy 5,8 dihydro 7- amino 5,8 dioxo 2 quinolyl) 4 (2 hydroxy- 3,4-dimethoxyphenyl)-3-methylpicolinic acid.

10. The methyl ester of the compound of claim 9.

11. The sodium salt of the compound of claim 9.

12. The calcium salt of the compound of claim 9.

13. The hydrazide of the compound of claim 9.

14. An antibiotic substance selected from the group consisting of streptonigrin F, a crystalline substance; which substance is weakly acidic, slightly soluble in water at neutral and acidic pHs and more soluble therein at alkaline pHs, very slightly soluble in ether, benzene, methylene chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethylacetate (1:4) and has a melting point of 185-187 C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 mu and 375-380 -m,u with Ei' g values of 850 and 370 respectively and, when measured in a KBr pellet containing 0.3% of the product; exhibits absorption maxima in the infrared region of the spectrum at 3410, 3330, 3250, 2920, 1735, 1680, 1630, 1600, 1584, 1564, 1478, 1440, 1400, 1370, 1339, 1270, 1230, 1208, 1180, 1162, 1100, 1092, 1071, 1050, 1036, 1008, 918, 873, 860, 828, 822, 806, 788, 760, 744, 702, 684 and 658 reciprocal centimeters; and which contains the elements carbon, hydrogen, nitrogen and oxygen in the following percentages by weight:

Carbon 60.54 Hydrogen 4.36 Nitrogen 11.42 Oxygen (by difference) 23.58

which substance provides a dark greenish-brown color solution with alcoholic ferric chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright-yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotically active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof.

15. An antibiotic substance selected from the group consisting of streptonigrin P a crystalline substance; which substance is weakly acidic, slightly soluble in water at neutral and acidic pHs and more solu-ble therein at alkaline pHs, very slightly soluble in ether, benzene, methylene chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethyl acetate (1:4) and has a melting point of 190-197" C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 my. (shoulder) and 375-380 mg with E}" values of 700 and 340 respectively 1023, 995, 919, 890, 868, 814, 804, 784, 746, 710, 682 and 652 reciprocal centimeters; and which contains the elements carbon, hydrogen, nitrogen and oxygen in the following percentages by weight:

20 Carbon 57.97 Hydrogen 4.72 Nitrogen 11.02 Oxygen (by difference) 26.29

which substance provides a dark greenish-brown colored solution with alcoholic ferric chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright-yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotically active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof.

16. An antibiotic substance selected from the group consisting of streptonigrin P a crystalline substance; which substance is weakly acidic, slightly soluble in water at neutral and acidic pHs and more soluble therein at alkaline pHs very slightly soluble in ether, benzene, methylene chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethylacetate (1:4) and has a melting point of 187-195 C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 mg and 375-380 m with Eifi values of 800 and 336, respectively and when measured in a KBr pellet containing 0.3% of the product exhibits absorption maxima in the infrared region of the spectrum at: 3320, 3210, 2900, 1715, 1675, 1630, 1608, 1570, 1555, 1500, 1460, 1440, 1405, 1375, 1342, 1280, 1228, 1184 1090, 1072, 1045, 1022, 995, 918, 890, 868, 814, 782, 747, 709, and 682 reciprocal centimeters; and which contains the elements carbon, hydrogen, nitrogen and oxygen in the following percentages by weight:

Carbon 58.12 Hydrogen 4.50 Nitrogen 10.60 Oxygen (by difference) 26.78

which substance provides a dark greenish-brown colored solution with alcoholic chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright-yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotically active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof.

17. An antibiotic composition comprising a substantial proportion of a non-toxic carrier and at least about 0.0001% of a product selected from the group consisting of the compounds of claim 8, the compounds of claim 14, the compounds of claim 15 and the compounds of claim 16.

18. A process for treating infections which comprises administering to a lower animal host a daily dose of about 50-500 mcg./ kg. of body weight of a compound selected from the group consisting of:

(a) 5 amino 6 (6 methoxy 5,8 dihydro 7- amino 5,8 dioxo 2 quinolyl) 4 (2 hydroxy- 3,4 dimethoxyphenyl) 3 methylpicolinic acid, the lower alkyl esters, the monomethyl ether, the hydrazide, the ammonium and the alkali metal and alkaline earth metal salts thereof,

(b) streptonigrin F, a cryst-alline substance; which substance is Weakly acidic, slightly soluble in water at neutral and acidic pHs and more soluble therein at alkaline pI-Is, very slightly soluble in ether, benzene, methylene chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethylace- 21 mm (1:4) and has a melting point of 185-187" C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 m and 375-3 80 11'l,u with EYZ values of 850 and 370, respectively and, when measured in a KBr pellet containing 0.3% of the product, exhibits absorption maxi-ma in the infrared region of the spectrum at 3410, 3330, 3250, 2920, 1735, 1680, 1630, 1600, 1584, 1564, 1478, 1440, 1400, 1370, 1339, 1270, 1230, 1208, 1180, 1162, 1100, 1092, 1071,1050, 1036, 1008, 918, 873, 860, 828, 822, 806, 788, 760, 744, 702, 684, and 658 reciprocal centimeters; and which contains the elements carbon, hydrogen, nitrogen and oxygen in the following percentages by weight:

Carbon 60.54 Hydrogen 4.36 Nitrogen 11.42 Oxygen (by difference) 23.58

which substance provides a dark greenish-brown color solution with alcoholic ferric chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright-yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotically active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof,

(c) streptonigrin P a crystalline substance; which substance is weakly acidic, slightly soluble in water at neutral and acidic pHs and more soluble therein at alkaline pHs, very slightly soluble in ether, benzene, methylene chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethyl acetate (1:4) and has a melting point of 190'197 C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 my (shoulder) and 375-380 m l. with EH13 values of 700 and 340, respectively and, when measured in a KBr pellet containing 0.3% of the product, exhibits absorption maxima in the infrared region of the spectrum at 3410, 3340, 3220, 2910, 2820, 1719', 1680, 1610, 1588, 1550, 1500, 1461, 1440, 1405, 1372, 1344, 1318, 1280, 1230, 11-86, 109 1, 1072, 1047, 1023, 995, 919, 890, 868, 814, 804, 784, 746, 710, 682, and 652 reciprocal centimeters; and which contains the elements carbon, hydrogen, nitrogen and oxygen in the following percentages by weight:

Carbon 57.97 Hydrogen 4.72 Nitrogen 11.02 Oxygen (by difference) 26.29

which substance provides a dark greenish-brown colored solution with alcoholic ferric chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotic-ally active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof,

(d) streptonigrin P a crystalline substance; which substance is weakly acidic, slightly soluble in water at neutral and acidic pHs and more soluble therein at alkaline pHs, very slightly soluble in ether benzene, methylene, chloride, and moderately soluble in ethyl acetate, the lower alcohols, and acetone; which crystallizes as red brown crystals from acetone-ethylacet-ate (1:4) and has a melting point of 187-195 C. (dec.); which substance exhibits absorption maxima in the ultraviolet region of the spectrum when dissolved in methanol at 245 my and 3753 mg with Eii' values of 800 and 356, respectively and when measured in a KBr pellet containing 0.3% f the product exhibits absorption maxima in the infrared region of the spectrum at: 3320, 3210, 2900, 1715, 1675, 1630, 1608, 1570, 1555, 1500, 1460, 1440, 1405, 1375, 1342, 1280, '1228, 1184, 1090, 1072, 1045, 1022, 995, 918, 890, 868, 814, 782, 747, 709, and 682 reciprocal centimeters; and which contains the elements carbon hydrogen, nitrogen and oxygen in the following percentages by weight:

Carbon 58.12 Hydrogen 4.50 Nitrogen i10.60 Oxygen (by difference) 26.78

which substance provides a dark greenish-brown colored solution with alcoholic chloride, dissolves in concentrated sulfuric acid to give a bright yellow solution, and the color of a dilute aqueous solution thereof changes from reddish-brown to bright yellow upon adding sodium bisulfite thereto, said bright yellow solution providing a reddish-brown antibiotically active extract on shaking with a water immiscible organic solvent; the lower alkyl esters, and the alkali metal and alkaline earth metal salts thereof.

References Cited UNITED STATES PATENTS 2,970,943 2/ 1961 Bromer et a1. 8O

FOREIGN PATENTS 61,749 12/ 1960 Australia. 1,089,928 9/1960 Germany.

848,936 6/ 1962 Great Britain.

OTHER REFERENCES Antibiotics & Chemotherapy, 1'1 (3), March 1961, pp. 147-189.

Rao et al., Antibiotics Annual, 1959-1960, pp. 950- 953, report of papers presented at the 7th Antibiotic Symposium, Wash., DC. Nov. 46, 1959.

ALBERT T. MEYERS, Primary Examiner.

FRANK CACCIAPAGLIA, JR., JULIAN S. LEVITT,

Examiners.

LEROY B. RANDALL, Assistant Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,372,090 March 5, 1968 William S. Marsh et a1.

It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

Columns 1 and 2, TABLE I, fifth column, line 3 thereof, for

"do" read None column 10, line 18, for "C H O N read C26H23O9N3 column 16, line 33, for "has" read gas column 18, line 26, for "fungicidus" read fungicidicus Signed and sealed this 22nd day of July 1969.

(SEAL) Attest:

Edward M. Fletcher, Jr. WILLIAM E. SCHUYLER, JR.

Attesting Officer Commissioner of Patents 

1. THE PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC CONTAINING PRODUCT WHICH COMPRISES CULTIVATING A STRAIN OF MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF 